Andrew Constanti

M-currents in voltage-clamped mammalian sympathetic neurones

The M-current (IM), a species of time- and voltage-dependent K+-current previously identified in amphibian sympathetic neurones, has been detected in voltage-clamped rat sympathetic neurone somata. IM had an activation threshold of about -70 mV and, since it did not show time-dependent inactivation, contributed an increasing outward component to the steady membrane current between -70 and -20 mV. Selective inhibition of IM by muscarine or by angiotensin-II probably accounts for the membrane depolarization produced by these two agonists on rat ganglia.

Differential effect of zinc on the vertebrate GABAA-receptor complex

1 γ‐Aminobutyric acid (GABA) responses were recorded from rat superior cervical ganglia (SCG) in culture using the whole cell recording technique. 2 Zinc (50–300 μm) reversibly antagonized the GABA response in embryonic and young post‐natal neurones, while neurones cultured from adult animals were far less sensitive and occasionally resistant to zinc blockade. Cadmium (100–300 μm) also antagonised the GABA response, while barium (100 μm‐2 mm) was ineffective. 3 The differential blocking effect of zinc on cultured neurones of different ages also occurred in intact SCG tissue. 4 The GABA log dose‐response curve constructed with foetal or adult cultured neurones was reduced in a non‐competitive manner by zinc. This inhibition was minimally affected by the membrane potential. 5 The GABA response recorded intracellularly from guinea‐pig pyriform cortical slices was enhanced by zinc (300–500 μm), which occurred concurrently with a decrease in the input conductance of the cell. The enhancement was unaffected by prior blockade of the GABA uptake carrier by 1 mm nipecotic acid. This phenomenon could be reproduced by barium (300 μm) and cadmium (300 μm). 6 We conclude that the vertebrate neuronal GABAA‐receptor becomes less sensitive to zinc with neural (GABAA‐receptor?) development, and the enhanced GABA response recorded in the CNS is a consequence of the reduction in the input conductance and not due to a direct effect on the receptor complex.

GABA receptors induced in Xenopus oocytes by chick brain mRNA: evaluation of TBPS as a use-dependent channel-blocker

Sensitivity to GABA was induced in Xenopus laevis oocytes by injection of poly(A)+ mRNA extracted from 19-day chick embryo brain. The effect of the convulsant drug t-butylbicyclophosphorothionate (TBPS) was studied on the responses to bath-applied GABA using voltage-clamp techniques. TBPS reversibly inhibited the GABA-evoked current (IGABA) in a dose-dependent manner; however, the chloride currents evoked by carbachol or serotonin, or the spontaneous chloride fluctuations, were unaffected. The onset of the block by TBPS was faster in the presence of GABA. The recovery from the block after TBPS wash-out was also agonist-stimulated. At the steady state block, TBPS showed a mixed type of inhibition: increasing the GABA concentration decreased but failed to abolish completely the inhibition by TBPS. The TBPS block was independent of the direction of the chloride flow: both inward and outward IGABA were blocked. The time course of the decay of IGABA was markedly changed in the presence of TBPS: above 40 microM GABA, this time course showed essentially two exponentials and TBPS abolished only the fast component, whereas at a low concentration (less than or equal to 4 microM), IGABA was relatively constant and uniformly reduced by TBPS. It is suggested that TBPS may stabilize a closed form of the liganded receptor-channel complex.

Studies on the Mechanism of Action of Picrotoxinin and Other Convulsants at the Crustacean Muscle GABA Receptor

The actions of picrotoxinin, bicuculline and penicillin-G were investigated on the GABA-receptor system of lobster muscle by using intracellular recording. The highly potent antagonist, picrotoxinin, produced a lateral shift and depression in the maximum of the GABA dose–conductance curve (designated as mixed antagonism); bicuculline, a weak antagonist, caused only a depression in the maximum with little or no lateral shift, whereas penicillin-G, an even weaker antagonist, produced a greater depression at the top of the dose–response curve. The possible sites of antagonist action were examined, with a critical re-evaluation of a drug-receptor model previously proposed to account for the antagonistic behaviour of picrotoxinin (the mixed antagonistic model); this model was extended to include the actions of bicuculline and penicillin-G. Antagonism was examined (i) towards different GABA receptor agonists; (ii) in various external anion media; (iii) at varying external pH; and (iv) when two different antagonists were combined. The GABA agonists were differentially antagonized by picrotoxinin and bicuculline, but external pH and substituent anions caused only minor perturbations to the inhibition. Combination experiments suggested at least three sites for GABA antagonists binding on crustacean muscle: (i) the GABA recognition site or sites; (ii) the ionic selectivity site in the ionophore; and (iii) a highly lipophilic site which may be part of the GABA receptor or ionophore. The mixed antagonism model accounted for the pH and external anion data but required modification to a cyclic scheme to explain the antagonism of a partial agonist. A model based on two-state receptor theory could only account for the antagonism of GABA if picrotoxinin was assumed not only to perturb L (the R ⇌ T conformation constant) but also to affect the agonist binding affinity. It is suggested that picrotoxinin and bicuculline may antagonize GABA responses by stabilizing the closed form of the activated channel, whereas penicillin-G may block the channel in the open state.

Nifedipine affects the anticonvulsant activity of topiramate in various animal models of epilepsy.

Topiramate (TPM), a new generation antiepileptic drug was investigated for its anticonvulsant effects in various models of genetically determined and chemically induced epilepsy in rodents. In addition, based on recent electrophysiological data suggesting that TPM may interact with L-type Ca(2+) channels, we evaluated the effects of a concomitant administration of L-type Ca(2+) channel modulators on TPMs antiepileptic properties. TPM, dose-dependently, protected against audiogenic seizures in DBA/2 mice. Concomitant treatment with TPM and a low dose of L-type Ca(2+) channel antagonists nifedipine or verapamil or with the L-type Ca(2+) channel agonist, S(-)-1,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)phenyl]-3-pyridinecarboxylic acid methyl ester (Bay k 8644) was able to increase the ED(50) for this drug. TPM also protected against seizures induced by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), 4-aminopyridine (4-AP) and pentylenetetrazole (PTZ), but this activity was not significantly modified by nifedipine. TPM, dose-dependently, reduced the number and duration of epileptic spike-wave discharges (SWDs) both in WAG/Rij rats and lethargic (lh/lh) mice, two genetic models of absence epilepsy. Nifedipine decreased TPMs activity in WAG/Rij rats but paradoxically enhanced it in lh/lh mice, whereas Bay k 8644 displayed opposite effects in both absence models. These results confirm TPMs broad spectrum of anticonvulsant activity and support the proposal that a modulation of neuronal L-type Ca(2+) channel activity plays an important role in its antiepileptic activity.

Medicinal cannabis: is Δ9–tetrahydrocannabinol necessary for all its effects?

Cannabis is under clinical investigation to assess its potential for medicinal use, but the question arises as to whether there is any advantage in using cannabis extracts compared with isolated Δ9‐trans‐tetrahydrocannabinol (Δ9THC), the major psychoactive component. We have compared the effect of a standardized cannabis extract (SCE) with pure Δ9THC, at matched concentrations of Δ9THC, and also with a Δ9THC‐free extract (Δ9THC‐free SCE), using two cannabinoid‐sensitive models, a mouse model of multiple sclerosis (MS), and an in‐vitro rat brain slice model of epilepsy. Whilst SCE inhibited spasticity in the mouse model of MS to a comparable level, it caused a more rapid onset of muscle relaxation, and a reduction in the time to maximum effect compared with Δ9THC alone. The Δ9THC‐free extract or cannabidiol (CBD) caused no inhibition of spasticity. However, in the in‐vitro epilepsy model, in which sustained epileptiform seizures were induced by the muscarinic receptor agonist oxotremorine‐M in immature rat piriform cortical brain slices, SCE was a more potent and again more rapidly‐acting anticonvulsant than isolated Δ9THC, but in this model, the Δ9THC‐free extract also exhibited anticonvulsant activity. Cannabidiol did not inhibit seizures, nor did it modulate the activity of Δ9THC in this model. Therefore, as far as some actions of cannabis were concerned (e.g. anti‐spasticity), Δ9THC was the active constituent, which might be modified by the presence of other components. However, for other effects (e.g. anticonvulsant properties) Δ9THC, although active, might not be necessary for the observed effect. Above all, these results demonstrated that not all of the therapeutic actions of cannabis herb might be due to the Δ9THC content.

Comparison of the antiepileptogenic effects of an early long-term treatment with ethosuximide or levetiracetam in a genetic animal model of absence epilepsy.

Purpose:  Epilepsy is a heterogeneous syndrome characterized by recurrent, spontaneous seizures; continuous medication is, therefore, necessary, even after the seizures have long been suppressed with antiepileptic drug (AED) treatments. The most disturbing issue is the inability of AEDs to provide a persistent cure, because these compounds generally suppress the occurrence of epileptic seizures without necessarily having antiepileptogenic properties. The aim of our experiments was to determine, in the WAG/Rij model of absence epilepsy, if early long‐term treatment with some established antiabsence drugs might prevent the development of seizures, and whether such an effect could be sustained.

A novel effect of zinc on the lobster muscle GABA receptor

The effect of zinc (and copper) was investigated on the lobster muscle γ-aminobutyric acid (GABA) receptor. Zinc (10 μm-1 mM) depressed the GABA-evoked conductance increase in a fully reversible manner by possibly binding to an imidazole group, suggested from pH titration studies on the evoked-chloride conductance. Other transition metal (period 4) divalent cations (up to 500 μM) were inactive in antagonizing GABA responses. Variation of external chloride or anion substitution did not perturb the zinc antagonism ; however, decreasing the pH markedly decreased the potency of zinc. A possible explanation for these results is discussed. Although the zinc antagonism resembled that produced by picrotoxinin, combination of these two agents depressed the GABA dose-conductance curve in a manner expected for two antagonists acting on independent sites. The zinc binding site was also discrete from the GABA recognition site; the results are interpreted in terms of a distinct binding site for zinc and H+. The distortion of an agonist dose-response curve by formation of an inactive agonist-divalent cation complex is discussed; however, complexation of GABA did not explain the observed antagonism by zinc. By comparison, zinc had no effect on the GABA responses of rat ganglionic neurons. It is concluded that the zinc binding site, on lobster muscle, may be an important modulatory site for the GABA-evoked chloride conductance.

The mTOR Signaling Pathway in the Brain: Focus on Epilepsy and Epileptogenesis

Recent evidence suggests that an altered mammalian (mechanistic) target of rapamycin (mTOR) signaling pathway and its pharmacological modulation might be implicated in several neurological diseases including epileptogenesis. mTOR is a molecular sensor, which regulates protein synthesis, enhancing mRNA translation of genes involved in the regulation of cell proliferation and survival, working as part of two distinct multimeric complexes known as mTORC1 and mTORC2. mTOR is an evolutionarily highly conserved serine/threonine kinase belonging to the phosphoinositide 3-kinase-related kinase family and represents one of the most recently studied pathways in relation to epilepsy and epileptogenesis, due to its suggested pivotal role in many aspects of cellular proliferation and growth also including neurodegeneration, neurogenesis, and synaptic plasticity. In this review, we report the cellular and molecular features of mTOR and related pathways, analyze their function in the brain including all current related evidence of their role, and finally, discuss the possible involvement of mTOR signaling in epileptogenesis and epilepsy, giving further consideration to future developments in this area.

Pre- and postsynaptic effects of zinc on in vitro prepyriform neurones

Intracellular recordings from guinea-pig olfactory cortex neurones revealed a dual effect of zinc: firstly (at 100-500 microM), the responses to bath-applied GABA, muscimol and 3-aminopropanesulphonate were reversibly enhanced, and secondly (at 25-500 microM), the excitatory postsynaptic potential was dramatically prolonged. At Zn2+ doses higher than 50 microM, transmission was eventually blocked irreversibly. These effects of zinc were not produced by 4-aminopyridine, or other divalent cations. The GABA-enhancement is suggested to occur via an interaction of Zn2+ with the post-synaptic GABA receptor, and the prolonged transmitter release probably via blockade of an outward current in nerve terminals. The latter effect may be a contributory factor in the epileptogenic activity of zinc.